Species specific DNA sequences and their utilization in identification of viola species and authentication of “banafsha” by polymerase chain reaction

ABSTRACT

This invention relates to  Viola  specific DNA sequences and methods for identification of  Viola  species using the said sequences. The invention also relates to a method for detection of adulteration in ‘banafsha’ by polymerase chain reaction method. Particularly this invention relates to a very sensitive, accurate and efficient method for identification of  Viola  species from even the powdered form of the plant or its admixtures with other plant species. More particularly, the method is designed to test the genuineness of ‘banafsha’.

FIELD OF THE INVENTION

The invention relates to Viola specific DNA sequences and methods foridentification of Viola species using the said sequences. The inventionalso relates to a method for detection of adulteration in ‘banafsha’ bypolymerase chain reaction method. Particularly this invention relates toa very sensitive, accurate and efficient method for identification ofViola species from even the powdered form of the plant or its admixtureswith other plant species. More particularly, the method is designed totest the genuineness of ‘banafsha’.

BACKGROUND OF THE INVENTION

Viola belonging to the family Violaceae is a genus of mostly perennialherbs. They are almost cosmopolitan but mostly distributed in thetemperate zones and restricted to the mountains in the tropics(Anonymous, 1965, Indian Pharmacopoeia, 2^(nd) Edn. Govt. of India;Anonymous, 1976, Wealth of India, PID, CSIR, New Delhi, Vol. X:514-517). There are about 400 species of Viola, of which about 30species are found in India. Viola species commonly known as “violets” or“pansies” hybridize freely in nature and, therefore, are difficult to bedistinguished taxonomically (Anonymous, 1976, Wealth of India, PID,CSIR, New Delhi, Vol. X: 514-517). The plants are available as differentcyto types with 2n=20, 37, 54 etc. in nature (Canne, J. M., 1987, CanadJ. Bot., 65; 653-655). Most of the Viola species yield perfumes andmedicaments, while some are ornamentals (Woodland, D. W., 1991,Contemporary plant systematics, Prentice Hall, Eaglewood, Cliffs). Themost important among these species, which is known for its medicinalsignificance in indigenous system of medicine, is Viola odorata L.,which is considered to constitute the genuine drug called “Banafsha”(Handa, K. L., Kapoor, L. D., Chopra, I. C. and Nath, S., 1951, IndianJ. Pharmacy, 13: 29-48). The whole plant is used in medicine and isavailable usually in 3 forms: 1) the dried aerial parts of the herb i.e.the stem, leaves and flowers; 2) only dried flowers and 3) aerial partswithout flowers. The plant is valued as sedative, expectorant,diaphoretic, antipyretic, diuretic, emetic, purgative, hypotensive andas a laxative (Kirtikar, K. R. and Basu, B. D., 1933, Indian MedicinalPlants, I: 205-212, Lalit Mohan Basu, Allahabad; Chopra, R. N., Nayer,S. L. and Chopra, I. C., 1956, Glossary of Indian Medicinal Plants,CSIR, New Delhi). It has also been reported to be antimycotic andantibacterial and is used for the treatment of eczema and inflammation.The flowers are credited with emollient and demulcent properties and areused as remedy for cough, sore throat, hoarseness, ailments of infants,billiousness and lung troubles and are also listed in FrenchPharmacopoeia (Lamaison, J. L., Petitjean, C. F. and Carnet, A., 1991,Plantes-Medicinales—et Phytotherapie, 25: 79-88). In Unani system ofmedicine, this plant is the main ingredient of “Joshanda” consisting ofmixtures of drugs and it is mostly used for cold, catarrhal, cough, andassociated fevers (Khetrapal, K., Khanna, T., Arora, R. B. and Siddiqui,H. H., 1987, Indian J. Pharmacy, 19: 200-204). The drug is prescribedmainly in the form of decoction, jam or syrup.

In India, Viola odorata wildly grows mainly in Jammu and Kashmir andoften cultivated elsewhere particularly in West Bengal, Gujarat, AndhraPradesh, Karnataka and Tamilnadu (Sharma, B. D., Balkrishan, N. P. 1993,Flora of India 2: 351-379). Due to the limited distribution and highdemand of Viola odorata, which is the main source of banafsha, alternatespecies including V. betonicifolia, V. biflora, Viola canescens, V.pilosa, V. sylvestris and V. tricolor (some of which grow abundantlythrough out the hilly regions of India) have been reported to besupplied in the market as banafsha, either in the pure form or aftermixing with V. odorata (Handa, K. L., Kapoor, L. D., Chopra, I. C. andNath, S., 1951, Indian J. Pharmacy, 13: 29-48: Dhar, et al, 1968, IndianJ. Exp. Biol., 6: 245; Mehrotra, S., Rawat, A. K. S. and Shome, U.,1998, Natural Plant Sci., 4: 14-22) which have led to the questionableefficacy of the drug, undermining the importance of Viola in medicine asoriginally defined (Kirtikar, K. R. and Basu, B. D., 1933, IndianMedicinal Plants, I; 205-212, Lalit Mohan Basu, Allahabad; Handa, K. L.,Kapoor, L. D., Chopra, I. C. and Nath, S., 1951, Indian J. Pharmacy, 13:29-48). It has also been reported that the roots of Viola odorata, V.cinerea and V. tricolor, which are emetic in nature, are also used assubstitute or adulterants of ipecac drug, which originally comes fromdried roots and rhizomes of Cephaelis ipecacuanha of family Rubiaceae(Ananymous, 1976, Wealth of India, PID, CSIR, New Delhi, Vol X: 514-517;Chopra, R. N., Nyer, S. L. and Chopra, I. C. Glossary of IndianMedicinal Plants, 1956, Edn. CSIR, New Delhi, 256). It is imperative,therefore, to identify the species composition of banafsha and otherherbal medicines before using them in various medicinal preparations,specially by the industrial houses. The classical ways of identifyingherbal medicines are based on the morphological, anatomical and chemicalanalysis using a variety of modern tools (Thankamma, A., Radhika, L. G.,1997, Aryavaidyan, XI: 52-56; Chauhan, S. K., Singh, B. P., Agarwal, S.,1995, Indian Drugs, 36: 189-191; Gawan, S. and Grampurohit, N. D., 1999,Indian Drugs, 36: 175-180). However, the structures and chemicalprofiles of the medicinal herbs are effected during sample processingand also by environmental and developmental factors during plant growth(Li, P., Pu, Z. M., Jiang, X., Liu, H. J., Xu, G. J., 1994, J. PlantRes. Envir., 3: 60-63; Cai, Z. H., Li, P., Dong, T. T. X. and Tsim, K.W. W., 1999, Planta Medica, 65: 360-364), for example, where marketsamples are in dry and powdered form, it is difficult to identify plantspecies by morphological characters. Similarly age, origin, harvestingperiod and the method of drying of plants may lead to the differences inthe secondary product formation and may interfere in identificationbased on chemical analysis.

Molecular markers are helpful in such situations, since they areindependent of environmental and developmental factors. They can beobtained even from dried and powdered herbs. RAPD markers have been usedto distinguish 3 different series of Viola tricolor (Oh, B. J., Ko, M.K., and Lee, C. H., 1998, Plant Breeding, 117: 295-296), to detectindividual component of Chinese medicinal prescription (Cheng, K., Tsay,H., Chen, C. and Chou, T., 1998, Planta Medica, 64: 563-565) and todetect dried roots of 3 Paax species and their adulterants (Shaw, P. C.and But, P. P. H., 1995, Planta Medica, 61: 466-469). However, thesemarkers are of little value specially in freely hybridizing populationsincluding Viola. Conserved sequences in such cases could be ideal. Infact, rRNA genes and their associated spacer length variability has beenutilized to differentiate fungal species (Nazar, R. N., Hu, X., Schmidt,J., Culham, D. and Robb, J., 1991, Physiol. and Mol. Plant Pathol., 39:1-11; Robb, J., Moukhamedov, R., Hu, X., plate, H. and Nazar, R. N.,1993, Physiol. and Mol. Plant Pathol. 43: 423-36; Moukhamedov, R. S.,Hu, X., Nazar, R. N. and Robb, J., 1994, Phytopathology, 83: 256-259),to identify plant varieties and species (Martsinkovskaya, A. I.,Moukhamedov, R. S. and Abdukarimov, A. A., 1996, Plant Mol. Biol.Reportr. 14: 44-49) and also to detect cereal composition in admixtures(Ko, H. L. and Henry, R. J., 1996, Plant Mol Biol. Reportr, 14: 33-43).Here, the applicants describe cloning and sequencing of the spacerregions between 5S rRNA genes in five Viola species, utilization of thesequence differences to detect the individual Viola species by a simplepolymerase chain reaction and to utilize this technology forauthentication of commercial samples of “banafsha”.

OBJECTS OF THE INVENTION

The main object of the invention is to provide Viola species specificDNA sequences.

Another object of the invention is to provide method for utilisation ofthe DNA sequences to identify Viola species.

Yet another object is to provide PCR based method for authentication ofthe composition of ‘banafsha’.

Still another object is to provide a method for detection ofadulteration of ‘banafsha’ and ‘ipecac’ drugs.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides Viola specific DNA sequences and methods foridentification of Viola species using the said sequences. The inventionalso provides a method for detection of adulteration in ‘banafsha’ bypolymerase chain reaction method. Particularly this invention relates toa very sensitive, accurate and efficient method for identification ofViola species from even the powdered form of the plant or its admixtureswith other plant species. More particularly, the method is designed totest the genuineness of ‘banafsha’.

BRIEF DESCRIPTION OF ACCOMPANYING FIGURES

FIG. 1: Diagrammatic illustration of 5SrRNA gene and their associatedspacer regions along with species specific primer combinations: M27 andM28 are forward and reverse consensus 5S rRNA gene primers respectively.BT61.F/BT61.R, BT811.F/BT811R, BT71.F/BT71.R, BT91.F and BT101.F are thespecific primers based on the DNA sequence of V. odorata, V. pilosa, V.canescens, V. betonicifolia and V. tricolor respectively. F and R areforward and reverse primers respectively. The expected amplificationproduct size in (base pairs) using either one consensus and the otherViola specific primer or both of the Viola specific primers are shown.The 5SrRNA coding region is ˜120 bps long.

FIG. 2: Species specific PCR products individually as well as inadmixtures: lanes 1 to 5 are V. odorata, V. canescens, V. pilosa, V.betonicifolia and V. tricolor respectively. Lane X is mixture of all the5 species DNA. B is blank lane without template DNA and M is mol. wt.marker e.g. 100 bp ladder. + and − shows presence or absence of theamplification product. Sets of primers used for the species shown on topof the gels were as illustrated in FIG. 1.

FIG. 3: PCR product obtained using V. pilosa or V. canescens specificprimers with admixtures of Viola species DNA minus V. pilosa or V.canescens: lanes 1 and 2 are with V. pilosa specific primers while lanes3 and 4 are with Viola canescens specific primers. Lanes 1 and 3 wereobtained with all DNA templates minus V. canescens while lanes 2 and 4have all DNA templates minus V. pilosa DNA. Lane B is blank lane with noDNA and M is mol. wt. marker 100 bp ladder.

FIG. 4: PCR products of 28 individual plants of Viola pilosa obtainedfrom 2 independent plots: lanes 1 to 28 represent individual plants. Mis mol. wt. marker λ HindIII/EcoRI double digest. Panel A shows PCRproducts using 5S rRNA gene consensus primers set M27 and M28 whilepanel B shows RAPD products of same plants using OPE-09 primer.

FIG. 5: Intra-species genetic homogeneity in Viola betonicifolia: lanes1 to 12 represent different plants. M is mol. wt. marker 100 bp ladder.Panel A1 shows PCR products using V. betonicifolia specific set ofprimers. Panel A2 is same PCR product after denaturation. Panel B showsRAPD patterns of the same plants using OPE-09 primer.

FIG. 6: Survey of Viola canescens in market samples of banafsha: Panel Ashows V. canescens specific primers. Lanes 1 to 18 represents samplesfrom various markets of India as listed serially in table 3. C ispositive control lane. M is mol. wt. marker 100-bp ladder.

DETAILED DESCRIPTION OF THE INVENTION

Accordingly, the present invention provides species specific DNAsequences and their utilization in identification of Viola species andauthentication of ‘banafsha’ by polymerase chain reaction, whichcomprises: a) unique sequences of 5 Viola species, b) unique set ofViola species specific PCR primers, c) a PCR based method to identifyViola species and d) a PCR based method to detect adulteration ofbanafsha even in dry powdered form.

In an embodiment of the present invention genomic DNA from 5 differentViola species was used (table 1).

TABLE 1 List of Viola species used in this work. S. No. Species name 1.Viola odorata 2. Viola pilosa 3. Viola canescens 4. Viola betonicfolia5. Viola tricolor

In another embodiment of the present invention genomic DNA from variousmarket samples of banafsha were used (table 2).

TABLE 2 List of various markets of India from which “banafsha” sampleswere obtained. S. No. Market 1. Srinagar (J & K) 2. Jammu (J & K) 3.Pathankot (Punjab) 4. Amritsar (Punjab) 5. Chamba (H.P.) 6. Shimla(H.P.) 7. Dharamshala (H.P.) 8. Jogindernagar (H.P.) 9. Kullu (H.P.) 10.Delhi (Khari baori) 11. Delhi (Karol bagh) 12. Lucknow (U.P.) 13.Dehradun* (U.P.) 14. Gwalior (Morar) 15. Hyderabad (A.P.) 16.Aurangabad$ 17. Aurangabad$ 18. Sholapur@ (Maharashtra) Note *Sarnimalbazar; $Pandariba road; @East mangaiwar road

In yet another embodiment of the present invention plasmid pMOS BlueT-vector DNA was used.

Genomic DNA extraction: The genomic DNA was isolated using the protocolof Saghai-Maroof et al. (Saghai-Maroof, M. A., Soliman, K. A.,Jorgenson, R. A. and Allard, R. W., 1984, Proc. Natl. Acad. Sci. (USA),81: 8014-18) as modified by us earlier (Singh, M., Sharma, C., andAhuja, P. S., 1999, Plant Mol Biol Reportr 17: 73).

PCR amplification, subcloning and sequencing of the amplified products:The consensus primers complementary to and based on the sequences of the3′ and 5′ ends of the 5S rRNA gene coding regions for plant as describedearlier (Kanazin, V., Ananiev, E. and Blake, T., 1993, Genome, 36:1023-28; Cox, V., Bennett, M. D. and Dyer, T. A., 1992, Theor ApplGenet, 83: 684-90) were got synthesized from “Bangalore Genei (Pvt)Ltd”, India. The sequences of these forward and reverse primers were(SEQ ID NO: 9) 5′-TITAGTGCTGGTATGATCGC-3′ (M27) and5′-TGGGAAGTCCTCGTGflTGCA-3′ (SEQ ID NO: 10) (M28) respectively. Theywere used to amplify the non coding spacer regions between 5S rRNAgenes. A 25 μl PCR reaction mix contained 2.5 μl of 1 of 10×PCR buffer,1 μl of dNTPs (stock of 2.5 mM each), 1 μl each of forward and reverseprimers (10 pico moles), 1.5 mM M_(g)Cl₂ and 0.5 units of Taqpolymerase.The PCR was performed in a Robocycler (Stratagene, La Jolla, Calif.)machine programmed for an initial heating at 94° C. for 3 min and thenfor 40 cycles at 94° C., 30 sec; 68° C., 30 sec and 72° C., 30 sec. Thefinal extension cycle at 72° C. was kept for 7 more minutes. The PCRproducts were analyzed in a 1.4% agarose gel and visualized in anethidium bromide staining under UV light. The lowest densely visiblebands in each of the five species were excised from the agarose gels andpurified using QIA quick gel extraction Kit (Qiagen). The purified DNAwas ligated in the pMOS Blue t-vector overnight at 16° C. as permanufacturers instruction (Amersham Life Sciences). The ligated mixturewas transformed into E.Coli MOS Blue cells and selected onto X-Gal, IPTGand ampicillin (100 μg/ml) containing Luria Bertani (LB) agar plates.Ten white colonies identified by colour selection were grown in 5 ml LBliquid medium overnight. The mini preparations of the plasmids wereperformed using alkaline lysis method (Sambrook, J., Fritsch, E. F. andManiatis, T., 1989, Molecular cloning: A laboratory manual. 2^(nd) Edn.,Cold Spring Harbour Laboratory Press, NY). The correct DNA inserts wereverified by restriction analysis. Finally one plasmid clone from eachplant was selected and its DNA prepared using Qiaquick plasmid DNAisolation Kit (Qiagen) which was then subjected to sequencing in boththe directions using the T7 promoter primers (SEQ ID NO: 11)(5′-TAATACGACTCACTATAGGG-3′) and M13 forward primer (SEQ ID NO: 12)(5′-CGCCAGGGTTTTCCCAGTCACGAC-3′) respectively on an applied Biosystemsmodel 377 automatic DNA sequencing system.

Sequence analysis and species specific primer design: The sequencingdata obtained from spacer regions between 5S rRNA genes were analyzedusing the PC Gene software. The complementary sequence from spacerregions specific to a particular Viola species which were dissimilar toother species were selected to be used as species specific primers.These primers are listed in table 3. The specific sets of primers usedto obtain a species specific PCR product and the sizes of the productsare illustrated in FIG. 1.

TABLE 3 Species specific primers used in this study SEQ ID NOS 1-8respectively, in order of appearance). S. Primer Total No code Sequencebases 1. BT61.F 5′---GGTGAGAACTCTCGAGGGTCGGG 24 A---3′ 2. BT61.R5′---GCCCCGATCCGACACCCGAGC---3′ 21 3. BT101.F5′---CCCTCACTCCTCGAGAATATG---3′ 21 4. BT91.F5′---CTATTTACTTCTCTCACCGCG---3′ 21 5. BT811.F5′---TTTGTAAACACGGAGGGGGC---3′ 20 6. BT811.R5′---ACAAACCCACGATTGGATTG---3′ 20 7. BT71.F5′---TTGTAAACACAGAGGAGGG---3′ 19 8. BT71.R 5′---CACGATTGGATTACACGC---3′18

In an embodiment BT 61.F and BT 61.R sequences when used as a primer,provides a single band as PCR product of 150 bps in the species Violaodorata.

In another embodiment BT 71.F and BT 71.R sequences when used as aprimer, provides a single band as PCR product of 200 bps in the speciesViola canescens.

In still another embodiment BT 811.F and BT 811.R sequences when used asa primer, provides a single band as PCR product of 200 bps in thespecies Viola pilosa.

In yet another embodiment BT 91F and M-28 sequences when used as aprimer, provides a single band as PCR product of 311 bps in the speciesViola betonicifolia.

In an embodiment BT 101.F and M-28 sequences when used as a primer,provides a single band as PCR product of 190 bps in the species Violatricolor.

In an embodiment the sequences specific to Viola species at an optimizedPCR annealing temperatures of 68° C. for V. odorata, 62° C. for V.canescens, 62° C. for V. pilosa, 52° C. for V.botonicifolia and 52° C.for V. tricolor.

In an embodiment, the sequences specific to Viola species at anoptimized PCR annealing time, said time being 15 second for V. odorata,24 second for V. canescens, 22 second for V. pilosa and 30 second for V.betonicifolia and V. tricolor respectively.

In yet another embodiment, the sequences specific to Viola species at anoptimized PCR extension time of 18 second for V. odorata, 17 second forV. canescens, V. pilosa, V. betonicifolia and V. tricolor respectively.

In yet another embodiment the sequences specific to Viola species at anoptimized concentration of MgCl₂ ions of 0.85 mM for V. odorata, 1.5 mMfor V. canescens, 1.1 mM for V. pilosa, 0.55 mM for V. betonicifolia and0.45 mM for V. tricolor respectively.

In yet another embodiment the sequences specific to Viola species at anoptimized primer concentration of 8-10 picomoles.

In yet another embodiment the sequences specific to Viola species givethe same PCR product with DNA isolated from Viola different plant partsi.e. from roots, rhizome and flowers.

The following examples are given by way of illustration of the presentinvention and should not be construed to limit the scope of the presentinvention.

EXAMPLE 1

Majority of the Viola Species Specific Primers Amplify OnlyCorresponding Species:

PCR amplification products using a set of primers, either one of thespecies specific and the other consensus or both species specificprimers (as illustrated in FIG. 1) were obtained for all five species.The PCR reaction contained 2.5 μl of 10×PCR buffer, 1 μl of dNTPs (mixof 2.5 mM each), 2-10 ng DNA, 8-10 picomole of each primer, 0.5 units ofTaq polymerase and a variable concentration (0.45-1.5 mM depending uponthe Viola species as shown in table 4) of MgCl₂ in a 25 μl reaction mix.The DNA was denatured at 94° C. for 3′ and then subjected to PCR upto 40cycles. A last extension cycle of 2 min in each case was also given at72° C. The annealing and extension temperatures and the times along withMgCl₂ concentration were optimized for each of 5 Viola species with eachset of primers (table 4) and are discussed below:

Viola odorata: Using 8 picomole each of the forward and reverse primers(BT61.F and BT61.R) respectively along with annealing at 68° C. for 15sec, extension time of 18 seconds and a concentration of 0.85 mM ofMgCl₂ was optimized to give a band of 150 bps as expected with only V.odorata but not with any of the other plants (FIG. 2, panel A, lane 1).There was also a similar amplification product when the genomic DNA ofall the five plant species were mixed in equal ratio (FIG. 2, panel A,lane X). The PCR product was V. odorata genome specific and could bedetected reproducibly in mixture of all the 5 plant species. There wasno significant difference in the band intensity when the genomic DNAfrom fresh tissues or the dried tissues were taken. Increasing MgCl₂concentration or the annealing time led to the appearance of a few faintbands of high molecular weight.

Viola canescens: Use of BT71.F and BT71.R as forward and reverse primersrespectively along with an annealing temperature of 62° C. for 24 secand an extension time of 17 sec along with a MgCl₂ concentration of 1.5mM gave an expected band of 200 bps with only V. canescens as well as inthe mixed samples of all 5 Viola species but not individually with anyof the other 4 DNA templates (FIG. 2, panel B, lanes 2 and Xrespectively). Increasing annealing temperature beyond 62° C. led to noamplification while reducing temperature led to appearance of severalfaint bands in addition to 1 prominent expected band.

Viola pilosa: Similarly use of BT811.F and BT811.R set of primers alongwith cycling parameters of annealing at 62° C. for 22 sec and extensionfor 17 sec along with a MgCl₂ concentration of 1.1 mM gave a band ofexpected size of 200 bps with V. pilosa and V. canescens but not withany of the other plants tested (FIG. 2, panel C, lanes 2 and 3). Similaramplification product was obtained when the genomic DNA of all the fiveplant species were mixed in equal ratio (lane X). To demonstrate that V.pilosa specific primers detect both V. pilosa and V. canescens but V.canescens specific primers detect only V. canescens specifically, theapplicants used species specific primers for these species to amplifymixtures of 4 Viola species DNA templates minus V. pilosa or V.canescens. As shown in FIG. 3 V. pilosa specific primers detect both V.pilosa and V. canescens but not vice versa.

Viola betonicifolia: Using BT91.F and M28 as forward and reverse primersrespectively along with an annealing temperature of 52° C. for 30 sec,an extension time of 17 sec, and MgCl₂ concentration of 0.55 mM gave anexpected band of 311 bps with only V. betonicifolia or in a mixture ofall 5 species but not with any of the other plants individually (FIG. 2,panel D, lanes 4 and X respectively).

Viola tricolor: Using BT101.F and M28 set of primers along with anannealing temperature of 52° C. for 30 sec, an extension time of 17 sec,along with a MgCl₂ concentration of 0.45 mM gave an expected band of 190bps with only V. tricolor or in a mixture of all 5 species but not withany of the other plants (FIG. 2, panel E, lanes 5 and X respectively).Surprisingly V. tricolor and V. betonicifolia required almost similaramplification conditions (Table 4).

TABLE 4 Standardized parameters for PCR amplification. S.N. ParameterV.O. V.P. V.C. V.B. V.T. 1. MgCl₂ (mM) 0.85 1.1 1.5 0.55 0.45 2.Annealing Temp (° C.) 68 62 62 52 52 Time (sec) 15 22 24 30 30 3.Extension time (sec) 18 17 17 17 17

EXAMPLE 2

Conserved 5S rRNA Gene Based Primers (3′ and 5′) Gave an IdenticalAmplification Product in a Population of 28 Individual Plants of Violapilosa:

In order to find out 5S rRNA associated spacer length and sequencevariability within the individuals of a species, the applicants analyzed28 individuals plants of V. pilosa selected randomly from 2 plots (fromIHBT campus, Palampur and Jogindernagar Herbal Garden, Jogindernagar),for their amplification products using consensus 5S rRNA primers M27 andM28. As shown in FIG. 4A, all the plants gave identical bandingpatterns. When these plants were subjected to RAPD analysis using OPE-09primer they again showed identical banding patterns (FIG. 4, panel B).When 2 more primers were analyzed they showed some differences in manyof the plants as expected ( data not shown).

EXAMPLE 3

Viola betonicifolia Specific Primers Gave an Identical AmplificationProduct in 12 Individual Plants Collected from Nature:

Similar to example 1, 12 individual plants of V. betonicifolia wereanalyzed for their amplification products using V. betonicifolia speciesspecific set of primers (BT91.F and M28 as illustrated in FIG. 2). Asshown in FIG. 5A1, all the plants gave identical banding patterns. ThesePCR products when denatured and analyzed in a 1.8% agarose gel againrevealed identical banding patters (FIG. 5, panel A2). When these plantswere subjected to RAPD analysis using OPE-09 primers they showed littledifferences, for example, in lane no 2 and 3 the 3^(rd) band from top ispresent only in 4 samples which is different in size in lane number 8(FIG. 5, panel B).

EXAMPLE 4

Market Samples of Banafsha Revealed that Majority of Them Were FakeSamples:

In order to find but the genuineness of banafsha, the applicantsanalyzed 18 market samples of banafsha collected from different marketsduring 1997-99 for their amplification products using V. canescensspecific primers. FIG. 6 shows that 7 samples were positive for V.canescens which does not represent genuine banafsha. This exampleclearly demonstrate that the developed PCR band approach works well formarket samples.

The main advantages of the present invention are:

-   1. It is specific to Viola odorata, V. pilosa, V. canescens, V.    betonicifolia and V. tricolor.-   2. It is highly sensitive and only nanogram amounts of DNA is    required.-   3. It can work equally well for degraded DNA.-   4. Only mg amounts of samples are required.-   5. It can work well for the processed and powdered samples.-   6. It can detect presence of Viola species even in admixtures of    samples and herbal formulations.-   7. The presence of Viola species specific PCR products can be    visualized in a simple agarose gel and no hazardous radioactive    labeling or time consuming and complex systems are needed.-   8. It is rapid.-   9. It has a potential for automation.

1. A method of detecting adulteration in samples of herb Viola odorataby other Viola species, said method comprising steps of: a. amplifyingthe sample DNA by PCR using a pair of primers selected from the groupconsisting of the following primer parts BT 71.F (SEQ ID NO: 7) andBT71.R (SEQ ID NO: 8); BT 811.F (SEQ ID NO: 5) and BT811.R (SEQ ID NO:6); BT 91.F (SEQ ID NO: 4) and M-28 (SEQ ID NO: 10); and BT 101.F (SEQID NO: 3) and M-28 (SEQ ID NO: 10); and b. detecting the adulteratedsample as the amplified sample.
 2. A method as claimed in claim 1,wherein the method is used to identify genuine Banafsha.